-
Science (New York, N.Y.) Oct 2023Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the...
Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.
Topics: Humans; Axonal Transport; Kinesins; Neurons; Synaptic Vesicles; Phosphatidylinositol Phosphates
PubMed: 37824668
DOI: 10.1126/science.adg1075 -
Cell Apr 2024In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional...
In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca and via its C2A domain-mediated Ca sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.
Topics: Animals; Synaptic Vesicles; COP-Coated Vesicles; Endoplasmic Reticulum; Calcium; Golgi Apparatus; Rats; Biological Transport; Presynaptic Terminals; Synapsins; Biomolecular Condensates; Cytoskeletal Proteins; Phase Separation
PubMed: 38552623
DOI: 10.1016/j.cell.2024.03.003 -
Proceedings of the National Academy of... Sep 2023The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis...
The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis is among the most serious diseases, but the mechanisms by which major meningitis-causing bacterial pathogens cross the BBB to reach the brain remain poorly understood. We found that , group B , and neonatal meningitis commonly exploit a unique vesicle fusion mechanism to hitchhike on transferrin receptor (TfR) transcytosis to cross the BBB and illustrated the details of this process in human BBB model in vitro and mouse model. Toll-like receptor signals emanating from bacteria-containing vesicles (BCVs) trigger K33-linked polyubiquitination at Lys168 and Lys181 of the innate immune regulator TRAF3 and then activate the formation of a protein complex containing the guanine nucleotide exchange factor RCC2, the small GTPase RalA and exocyst subcomplex I (SC I) on BCVs. The distinct function of SEC6 in SC I, interacting directly with RalA on BCVs and the SNARE protein SNAP23 on TfR vesicles, tethers these two vesicles and initiates the fusion. Our results reveal that innate immunity triggers a unique modification of TRAF3 and the formation of the HBMEC-specific protein complex on BCVs to authenticate the precise recognition and selection of TfR vesicles to fuse with and facilitate bacterial penetration of the BBB.
Topics: Humans; Animals; Mice; Infant, Newborn; Blood-Brain Barrier; Endothelial Cells; TNF Receptor-Associated Factor 3; Transcytosis; Bacteria; Receptors, Transferrin
PubMed: 37733740
DOI: 10.1073/pnas.2307899120 -
Journal of Extracellular Vesicles Jan 2024Blood is the most commonly used body fluid for obtaining and studying extracellular vesicles (EVs). While blood is a standard choice for clinical analysis, using blood... (Review)
Review
Blood is the most commonly used body fluid for obtaining and studying extracellular vesicles (EVs). While blood is a standard choice for clinical analysis, using blood as a source of EVs introduces multiple layers of complexity. At the Blood Extracellular Vesicle Workshop organized by the International Society for Extracellular Vesicles in Helsinki (2022), it became evident that beginner researchers lack trustworthy information on how to initiate their research and avoid common pitfalls. This educational guide explains the composition and frequently used terminology of blood, provides guidelines for blood collection, and the preparation of plasma and serum. It also introduces the basic principles of isolating and detecting blood EVs while considering blood-related factors. The goal of this guide is to assist beginners by offering a concise and evidence-based introduction to the current knowledge and available resources to study blood EVs.
Topics: Humans; Extracellular Vesicles; Plasma; Body Fluids
PubMed: 38193375
DOI: 10.1002/jev2.12400 -
Nano Letters Jan 2024Extracellular vesicles and lipoproteins are lipid-based biological nanoparticles that play important roles in (patho)physiology. Recent evidence suggests that... (Review)
Review
Extracellular vesicles and lipoproteins are lipid-based biological nanoparticles that play important roles in (patho)physiology. Recent evidence suggests that extracellular vesicles and lipoproteins can interact to form functional complexes. Such complexes have been observed in biofluids from healthy human donors and in various disease models such as breast cancer and hepatitis C infection. Lipoprotein components can also form part of the biomolecular corona that surrounds extracellular vesicles and contributes to biological identity. Potential mechanisms and the functional relevance of extracellular vesicle-lipoprotein complexes remain poorly understood. This Review addresses the current knowledge of the extracellular vesicle-lipoprotein interface while drawing on pre-existing knowledge of liposome interactions with biological nanoparticles. There is an urgent need for further research on the lipoprotein-extracellular vesicle interface, which could return important mechanistic, therapeutic, and diagnostic findings.
Topics: Humans; Lipoproteins; Extracellular Vesicles
PubMed: 38122812
DOI: 10.1021/acs.nanolett.3c03579 -
ACS Nano Sep 2023Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that...
Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.
Topics: Animals; Mice; Glioblastoma; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Glioma; Extracellular Vesicles; Glutathione
PubMed: 37646615
DOI: 10.1021/acsnano.2c12857 -
Current Opinion in Cell Biology Aug 2023The compartmentalization of eukaryotic cells is reliant on the fidelity of vesicle-mediated intracellular transport. Vesicles deliver their cargo via membrane fusion, a... (Review)
Review
The compartmentalization of eukaryotic cells is reliant on the fidelity of vesicle-mediated intracellular transport. Vesicles deliver their cargo via membrane fusion, a process requiring membrane tethers, Sec1/Munc18 (SM) proteins, and SNAREs. These components function in concert to ensure that membrane fusion is efficient and accurate, but the mechanisms underlying their cooperative action are still in many respects mysterious. In this brief review, we highlight recent progress toward a more integrative understanding of the vesicle fusion machinery. We focus particular attention on cryo-electron microscopy structures of intact multisubunit tethers in complex with SNAREs or SM proteins, as well as a structure of an SM protein bound to multiple SNAREs. The insights gained from this work emphasize the advantages of studying the fusion machinery intact and in context.
Topics: Membrane Fusion; Cryoelectron Microscopy; SNARE Proteins; Munc18 Proteins
PubMed: 37421936
DOI: 10.1016/j.ceb.2023.102191 -
Trends in Endocrinology and Metabolism:... Sep 2023The body partially maintains metabolic homeostasis through interorgan communication between metabolic organs under physiological conditions. This crosstalk is known to... (Review)
Review
The body partially maintains metabolic homeostasis through interorgan communication between metabolic organs under physiological conditions. This crosstalk is known to be mediated by hormones or metabolites, and has recently been expanding to include extracellular vesicles (EVs). EVs participate in interorgan communication under physiological and pathological conditions by encapsulating various bioactive cargoes, including proteins, metabolites, and nucleic acids. In this review we summarize the latest findings about the metabolic regulation of EV biogenesis, secretion, and components, and highlight the biological role of EV cargoes in interorgan communication in cancer, obesity, diabetes, and cardiovascular disease. We also discuss the potential application of EVs as diagnostic markers, and corresponding therapeutic strategies by EV engineering for both early detection and treatment of metabolic disorders.
Topics: Humans; Extracellular Vesicles; Metabolic Diseases; Neoplasms; Cell Communication; Communication
PubMed: 37394346
DOI: 10.1016/j.tem.2023.06.002 -
Journal of Controlled Release :... Sep 2023Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells. EVs are emerging as a promising class of therapeutic entity that could be adapted in... (Review)
Review
Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells. EVs are emerging as a promising class of therapeutic entity that could be adapted in formulation due to their lack of immunogenicity and targeting capabilities. EVs have been shown to have similar regenerative and therapeutic effects to their parental cells and also have potential in disease diagnosis. To improve the therapeutic potential of EVs, researchers have developed various strategies for modifying them, including genetic engineering and chemical modifications which have been examined to confer target specificity and prevent rapid clearance after systematic injection. Formulation efforts have focused on utilising hydrogel and nano-formulation strategies to increase the persistence of EV localisation in a specific tissue or organ. Researchers have also used biomaterials or bioscaffolds to deliver EVs directly to disease sites and prolong EV release and exposure. This review provides an in-depth examination of the material design of EV delivery systems, highlighting the impact of the material properties on the molecular interactions and the maintenance of EV stability and function. The various characteristics of materials designed to regulate the stability, release rate and biodistribution of EVs are described. Other aspects of material design, including modification methods to improve the targeting of EVs, are also discussed. This review aims to offer an understanding of the strategies for designing EV delivery systems, and how they can be formulated to make the transition from laboratory research to clinical use.
Topics: Tissue Distribution; Extracellular Vesicles
PubMed: 37536545
DOI: 10.1016/j.jconrel.2023.07.059 -
Journal of Extracellular Vesicles Aug 2023Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system...
Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain-derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size-exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG-UC). Our results showed that SG-UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass-tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG-UC showing the best enrichment of common EV proteins with less contamination of non-EV proteins. In addition, SG-UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell-substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single-particle analysis using the super-resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.
Topics: Humans; Aged; Extracellular Vesicles; Proteomics; Lipoproteins; Microscopy, Electron, Transmission; Brain
PubMed: 37563857
DOI: 10.1002/jev2.12358